Consistent Errors in First Strand cDNA Due to Random Hexamer Mispriming

T.P. van Gurp, L.M. McIntyre, K.J.F. Verhoeven

Research output: Contribution to journalArticleAcademicpeer-review

21 Citations (Scopus)

Abstract

Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.
Original languageEnglish
Article numbere85583
Number of pages4
JournalPLoS ONE
Volume8
Issue number12
DOIs
Publication statusPublished - 2013

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Complementary DNA
RNA
Nucleotides
nucleotides
Research Design
synthesis
DNA
Datasets

Keywords

  • dna-sequence differences
  • human transcriptome
  • widespread rna
  • discovery
  • biases

Cite this

van Gurp, T. P., McIntyre, L. M., & Verhoeven, K. J. F. (2013). Consistent Errors in First Strand cDNA Due to Random Hexamer Mispriming. PLoS ONE, 8(12), [e85583]. https://doi.org/10.1371/journal.pone.0085583
van Gurp, T.P. ; McIntyre, L.M. ; Verhoeven, K.J.F. / Consistent Errors in First Strand cDNA Due to Random Hexamer Mispriming. In: PLoS ONE. 2013 ; Vol. 8, No. 12.
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Consistent Errors in First Strand cDNA Due to Random Hexamer Mispriming. / van Gurp, T.P.; McIntyre, L.M.; Verhoeven, K.J.F.

In: PLoS ONE, Vol. 8, No. 12, e85583, 2013.

Research output: Contribution to journalArticleAcademicpeer-review

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